Metal Coating of Rare Earth Nano-Phosphors and Uses Thereof

ABSTRACT

Core-shell nanoparticles comprises a phosphorescent core and metal shell comprising at least two metals The phosphorescent core may comprise an up converting phosphor. The phosphorescent core may comprise a trivalent rare earth cation. The phosphorescent core further may comprise a monovalent alkali metal. The phosphorescent core may optionally comprises a second and also optionally a third trivalent rare earth cation.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.2009-35603-05070, awarded by the United States Department ofAgriculture. The Government has certain rights in this invention.

SUMMARY OF THE INVENTION

A nanoparticle comprising a phosphorescent core and a metal shellcomprising at least two metals is provided. In one embodiment, thephosphorescent core comprises an upconverting phosphor. In anotherembodiment, the phosphorescent core comprises a trivalent rare earthcation. In one embodiment, the phosphorescent core further comprises amonovalent alkali metal. The phosphorescent core optionally comprises asecond and also optionally a third trivalent rare earth cation.

In one embodiment, the rare earth cation is Tm³⁺, Er³⁺, Y³⁺, or Yb³⁺.

In another embodiment, the phosphorescent core further comprises a Group15, 16 or 17 anion. The Group 17 anion is F⁻ in some embodiments.

In one embodiment the monovalent alkali metal is Na⁺.

In one embodiment the at least two metals are transition metals. Inanother embodiment, the at least two metals are selected from the groupconsisting of Au, Ag, Pt, Pd, Rh and Re.

In one embodiment, nanoparticle further comprises a fluorescent tag.

In one embodiment, the nanoparticle further comprises a magneticcomponent.

In one embodiment the phosphorescent core comprises the magneticcomponent.

In one embodiment the magnetic component is magnetite (Fe₃O₄).

Also provided are methods of incorporating the nanoparticles intosecurity paper.

Also provided is a security paper comprising a plurality ofnanoparticles wherein said nanoparticles are visible and of a firstcolor and upon excitation with a low-powered laser, said nanoparticlesemit a second color.

In one embodiment, the second color emitted upon excitation is red, blueor green, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the synthesis of gold-coated phosphor CSNP.

FIG. 2 is an absorbance spectrum of 0.1% solution of gold-coatedphosphor CSNP.

FIG. 3 is a transmission electron micrograph image of gold-coatedphosphor CSNP heated to 450° C.

FIG. 4 are HAADF-STEM micrographs of gold-coated and uncoated phosphorCSNPs.

FIG. 5 is an additional HAADF-STEM image for gold-coated phosphor CSNPswith accompanying EDS line spectra.

FIG. 6 are additional HAADF-STEM images for gold-coated phosphor CSNPswith accompanying EDS line spectra.

FIG. 7 are X-ray diffraction and optical absorbance measurements forgold-coated and uncoated phosphor CSNPs.

FIG. 8 are bright field TEM micrograph images for heated gold-coatedphosphor CSNPs and X-ray diffraction data.

FIG. 9 are particle size distributions from dynamic light scatteringexperiments.

FIG. 10 shows absorbance and up-conversion emission spectra forgold-coated and uncoated phosphor CSNPs.

FIG. 11 shows absorbance and up-conversion emission spectra forgold-coated and uncoated phosphor CSNPs.

FIG. 12 shows up-conversion emission spectra for gold-coated anduncoated phosphor CSNPs.

FIG. 13 illustrates the process of labelling gold-coated phosphor CSNPwith streptavidin-tetramethylrhodamine (SA-TMR).

FIG. 14 is an absorbance spectra showing the result of conjugationSA-TMR to gold-coated phosphor CSNP as described in Example 3.

FIG. 15 is an emission spectrum comparing gold-coated phosphor CSNPalone, gold-coated phosphor CSNP conjugated to streptavidin (SA) andgold-coated phosphor CSNP conjugated to SA-TMR as described in Example3.

FIG. 16 is an emission spectrum of ALEXA FLUOR™ tagged gold-coatedphosphor CSNP excited with 980 nm light.

FIG. 17 are bright-field transmission electron micrographs of magnetitecontaining gold-coated phosphor CSNPs.

FIG. 18 are bright-field transmission electron micrographs of magnetitecontaining uncoated phosphor CSNPs.

FIG. 19 is a graph of superparamagnetic hysteresis measurements ofgold-coated and uncoated phosphor CSNPs with magnetite cores.

FIG. 20 shows absorbance spectra of three different solutions ofNaYF₄:20% Yb:2% Er containing different molar ratios of Ag and Au.

DETAILED DESCRIPTION

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. The practice ofthe present invention will employ, unless otherwise indicated,conventional methods of chemistry and spectroscopy.

The rare earth elements are the lanthanide series, scandium (Sc) oryttrium (Y). The lanthanide elements are lanthanum (La), cerium (Ce),praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm),europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium(Ho), erbium (Er), thulium (Tm), ytterbium (Yb) and lutetium (Lu).

A rare earth cation is the positive ion of one of the rare earthelements.

A transition metal is any element which has an incomplete d sub-shell orcan give rise to cations with an incomplete d sub-shell.

A metal precursor is a compound that leads to the elemental metal in oneor more reactions.

Numbering of the groups of the periodic table is according to the IUPACrecommendation of numbering groups of the periodic table with numbers1-18.

An upconverting phosphor is a phosphor which emits radiation at awavelength that is shorter than the excitation wavelength.

Förster Resonance Energy Transfer (FRET)

One of the most common optical detection techniques used for bioassaysis fluorescence detection. Some of these techniques use an affinitybased energy transfer phenomenon called Förster Resonance EnergyTransfer (FRET) (sometimes referred to as Fluorescence Resonance EnergyTransfer). A non-radiative energy transfer occurs between a donor and anacceptor depending on the distance between them and the overlap of theemission spectrum of the donor and the excitation spectrum of theacceptor. The transfer efficiency depends inversely on the sixth powerof the distance between the donor and the acceptor molecule leading to apowerful nanoscale (2-10 nm) measurement technique. The transferefficiencies for a wide variety of fluorescent dyes have been studied.Non-fluorescent quenchers such as QSY can also be used as efficientacceptors (donors) because of their availability in a wide spectrumrange and the absence of any intrinsic fluorescence emission. FRET canbe used as an efficient detection tool for fluoroimmunoassayapplications. Antibody and antigen pairs can be tagged with FRET pairs,e.g., antibody with a fluorescent dye and antigen with its FRET pair;and used in different immunoassay formats including competitive assays.The binding reaction can be observed as a simultaneous decrease in thedonor signal and an increase in the acceptor signal due to the energytransfer between the fluorescent dyes when they are in close proximity.If a non-fluorescent dye (quencher) is used as an acceptor, then onlythe decrease in the donor signal is observed relative to the reactionbetween the antibody-antigen.

A CSNP is one example of a biomarker that can be utilized with FRET. Thecore of the CSNP is a phosphor particle and the shell coating the coreis metal. Rare earth, or lanthanide, cations are one example of aphosphor particle core that can be used with the teachings describedherein. Rare earth ion phosphors are readily synthesized in differentsizes, shapes and colors. Rare earth ions have an advantage over quantumdots as biomarkers, since some of the ions emit in the visible region(blue from Thulium, Tm³⁺; green and red from Erbium, Er³⁺) when excitedby a near infrared light[1-3]. This phenomenon known as upconversion isnot only inexpensive, but well-suited for labeling applications. Lowerexcitation energy of the phosphor eliminates auto fluorescence from mostproteins, thereby eliminating unwarranted interference in quantificationof interaction when the CSNP is used in bioassays. Förster resonanceenergy transfer (FRET) is a simple technique to detect interactionbetween biomolecules over a short range through the non-radiativetransfer of energy from a donor to an acceptor molecule. Rare earthphosphor nanoparticles and organic fluorescent tags attached to DNA orprotein may be an attractive system for FRET since a wide variety ofnanoparticles is easily synthesized [4-6].

Metal nanoparticles are used in FRET based assays as promoters ofresonance energy transfer between fluorophores [7]. The enhancement orsuppression of fluorescence by the metal is dependent on the shape,size, distance and orientation of the nanoparticle [7-11]. Plasmonic anddielectric materials in a core-shell architecture can be employed toutilize the plasmonic enhancement of the fluorescence emission in a FRETconfiguration [9, 12].

Using two metals in the shell provides the opportunity for tuning ofproperties of phosphor CSNPs for particular applications. Adding thesecond metal can cause the shift in the plasmon absorption peak. Forexample, nanoparticles of gold have plasmon absorption peaks that arepredominantly observed at wavelengths ≧500 nm. Adding silver to goldchanges the absorption peak to wavelengths ≦500 nm. The absorption peakshifts to higher wavelengths with increasing metal nanoparticle size orthe core size on which they are coated. One can understand that usingtwo metals, 1) the color of the phosphor CSNPs can be tuned from blue toNIR. 2) the color of the phosphor CSNPs can be made the same fordifferent phosphor core sizes by a suitable choice of the two metals,their size and amounts. The changes in the composition of the metalsinfluences the absorption and emission characteristics of the phosphorcore. Apart from the plasmonic effects of the metal, the crystal fieldsaround activators and emitters are influenced by phonons. The type ofmetal, their size and amount, in and around the phosphor selectivelyaffects the different emissions from the phosphor to generate a specificcolor. Incorporation of silver as the second metal adds antifungal andantibacterial properties to the phosphor CSNPs. Since silver has a lowermelting point, sintering of phosphor CSNPs can be achieved at lowertemperatures. The sintered phosphor CSNPs will result in new ceramic(dielectric)-metal compositions (composites) with unique optical andconductive properties.

In one embodiment, the CSNPs include magnetic or super-paramagneticparticles. This component provides a handle to manipulate the particleand can also play a role as a functional adduct in providing a magneticsignal in immunoassays when used as sensors. Further, they allowmanipulating and sorting particles or analytes in micro- or nano-fluidicdevices. In embodiments where the CSNP is used for magnetic hyperthermiatherapy the magnetic component is useful in heat generation. Magneticparticles for use with the disclosed gold-coated phosphor CSNPs includemagnetite (Fe3O4), rare earth elements including Nd, Sm and Gd andalloys including Fe, B or other non-lanthanide elements.

EXAMPLES Example 1 Preparation and Characterization of Gold-Coated CSNPs

FIG. 1 illustrates the synthesis of the gold-coated phosphor CSNP. InFIG. 1, Ln³⁺ is the representative rare earth trivalent cation. The CSNPis prepared by a solution based technique. A 0.2 M solution of YCl₃ (78%by molecular weight), YbCl₃ (20%) and ErCl₃ (2%) was mixed with 0.2 Msodium citrate in a 1:2 volume ratio and heated to 90° C. 1 M NaF wasadded at four times the volume of the solution forming a white coloredsolution. Then 380 nmoles of 0.1% HAuCl₄ were added and the heating wascontinued for two and half hours resulting in gold-coated NaYF₄nanoparticles containing Yb³⁺ and Er³⁺ that were pink in color. Thenanoparticles were centrifuged and dried at 80-100° C. resulting in axerogel. The xerogel was crushed and heated to 450° C. for 12 hoursunder continuous nitrogen flow in a custom built tubular furnace.

The resulting gold-coated phosphor CSNPs were characterized by creatinga 0.1% solution in water. The absorbance was measured by a SPECTRAMAX™M2 from Molecular Devices in Sunnyvale, Calif. The results are shown inFIG. 2. The solid line represents absorbance of the gold coated CSNPsand the dashed line represents absorbance of the nanoparticle prior tothe gold coating. The discontinuity in the absorbance of the gold coatedCSNP between 500 and 600 nm arises from the gold surface plasmonresonance providing evidence that the gold shell has been deposited.

Additionally the CSNPs were heated to 450° C. and imaged by transmissionelectron microscopy (CM-12 scanning transmission electron microscopefrom Philips in Eindhoven, Netherlands). FIG. 3 is the transmissionelectron micrograph. The arrows point to the thin layer of gold of 4-8nm surrounding the phosphor. These nanoparticles displayed green and redcolor emissions when excited by 975-980 nm operated at an average powerof 22 mW (±0.5 mW).

Example 2 Additional Nanoparticles

A 0.2 M solution of YCl₃ (78% by molecular weight), YbCl₃ (20%) andErCl₃ (2%) was mixed with 0.2 M sodium citrate and 1 M NaF (SigmaAldrich) solution in a 1:2:4 volume ratio and heated to 90° C. Then 1.52μmoles of 0.1% HAuCl₄ (Alfa Aesar) were added to the white coloredsolution, and the heating was continued for two and half hours. Thesynthesis of core nanophosphor (without gold coating) was done with thesame above procedure without the addition of the HAuCl₄, resulting inwhite colored nanoparticles. Both sets of nanoparticles were centrifugedand dried at 80-100° C. The resulting xerogel was crushed and heated to350° C. for 12 h in N2 flow furnace.

Transmission electron microscopy in which strong atomic-number (Z)contrast verifies the presence of gold was used to analyze the resultingnanoparticles. In Z-contrast imaging, the contrast in the image isdependent on the atomic number of the observed atom with intensitiesroughly proportional to Z2.[13] The powder samples were deposited on aformvar/carbon coated copper grids from a water solution, dried beforetaking the bright field transmission electron micrographs from PhillipsCM-12 operating at 120 KeV and 15 mA. The HAADF-STEM images were takenusing a field emission gun JEOL JEM 2500SE (S)TEM instrument operated at200 kV with a 1 nm spot size and 800 mm camera length, corresponding toan inner and outer semi-collection angle of 35 and 90 mrad,respectively. FIG. 4 shows the Z-contrast images from scanningtransmission electron microscopy (STEM). FIGS. 4 a and 4 b show theuncoated NaYF₄:20% Yb:2% Er nanophosphors. FIGS. 4 c and 4 d show thecore-shell architecture of the gold-coated nanophosphors. In FIGS. 4 cand 4 d, the gold shell was visible on top of the NP core. Due to theatomic number difference between the yttrium (Z=39) core and the gold(Z=79), the gold shell appears as a ‘bright ring’ on top of the core.This was further verified by imaging the uncoated NP in the same mode(FIGS. 4 a and 4 b). Such bright features were absent—confirming theformation of gold coated NPs. The particle sizes of both coated anduncoated NPs were similar at about 20-50 nm; the particles appeared tobe porous in nature. From the bright field TEM micrographs, no secondarynanoparticle phase that could be attributed to the gold nanoparticles isvisible, even as the gold precursor was increased stepwise in thesolution. This can be attributed to the fact that the reductant citrateion is not present in the solution but is confined to the surface of thecore NP—the nucleation rate in the solution is minimized, and isenhanced on the surface of the core.14 Optimization of the gold coatingmethod to avoid nucleation of the gold nanoparticles was achieved byvarying the concentration of the HAuCl₄ solution. A dilute solution of0.1% eliminated the growth of Au nanoparticles (FIG. 4 f) on the surfaceand enhanced the formation of the shell when compared to 1% solution(FIG. 4 e).

FIGS. 4 c and 4 d show continuous gold shells of 4-8 nm achieved usingthe highest concentration of 0.1% HAuCl₄ precursor in the solution,corresponding to Au/Tm and Au/Er of 0.19 and 0.095. There is minimalvariation across various nanoparticles as demonstrated by the EDSspectra in FIG. 5. EDS spectra in FIG. 5 from different nanoparticlesprovide a semi-quantitative confirmation of the Au shell uniformity,where the ratio of Au to Y was ˜0.07 when integrated area under thecurves are considered (See also FIG. 6 of additional HAADF-STEM imagesand EDS spectra for gold-coated phosphor CSNPs).

Powder X-ray Diffraction

The phases of the calcined powders of gold-coated and uncoated phosphorCSNPs were characterized by Sintag XDS 2000 fitted with a copper Kasource, operating at −45KV and 40 mA. Powdered samples were spread on alow background glass slide and scanned in a 2θ-2θ geometry from 10 to 60degrees. FIG. 7 a shows X-ray diffraction data for both gold-coated anduncoated cubic α-NaYF₄ (JCPDS-77-2042) nanophosphors containing Er³⁺ andTm³⁺ heated to 90° C. for 12 h. The presence of the Au shell was notdetected from X-ray diffraction and it appears that the Au shell isamorphous. Even the heating of the gold-coated phosphor CSNPs to 350° C.did not result in the crystallization of the Au shell (FIG. 8 b).However, the NaYF₄:Yb:Er/Tm was cubic α phase when synthesized and driedat 90 C (FIG. 7 a).

The gold-coated and the uncoated phosphor CSNPs (NaYF₄:20% Yb:2% Er andNaYF₄:20% Yb:1% Tm) were heated to 350° C. for four hours in a tubularfurnace under a controlled nitrogen atmosphere. To maintain the sameheat treatment all samples were heated in a batch, thus subjecting bothto the same conditions. The X-ray diffraction (FIG. 8 d) of the heatedsamples show that the phase of the nanoparticle is a mixture of α(JCPDS-77-2042) and β JCPDS-39-0724) NaYF₄ phases, with β NaYF₄ beingthe dominant phase. The effect of heat treatment can be quantified fromthe ratio of X-ray intensities arising from the (220) and (111) planes,corresponding to 100% intensities of α and β NaYF₄ phases, respectively.In the coated and uncoated cases, the ratio of intensity,I(220)α/I(111)β, was 3.5 and 3.2 respectively. Given the inhomogeneousdistribution of the two phases in the sample, and the error in theamount of sample exposed to X-rays, the two phases in the coated and theuncoated can be considered to be effectively equal.

A distinct shell of 4-8 nm is visible in the bright field TEM micrographof the calcined gold coated NP (FIG. 8 a-c; FIGS. 8 a and 8 b areNaYF₄:20% Yb:2% Er with Au/Er ratio of 0.095 with 8 a heated to 90° C.for 12 hours and 8 b heated to 350° C. for four hours; FIG. 8 c isNaYF₄:20% Yb:2% Er with Au/Er ration of 0.285 heated to 350° C. for 12hours).

Absorbance and Emission Experiments

The absorbance and emission experiments were performed with 0.1%solution of the colloids prepared using cubic α-NaYF4:20% Yb:1% Tm andcubic α-NaYF4:20% Yb:2% Er nanophosphors dried at 90° C. for 12 h. TheCSNPs had varying ratios of Au to Tm/Er as shown on the graphs of FIG.9. The colloids were characterized by dynamic light scatteringexperiments (Brookhaven ZetaPlus) for aggregation. A single run was oftwo minutes duration and the data was averaged over five runs for eachsample. The colloidal suspension has a particle size distributioncorresponding to the primary particle size of the nanophosphors (FIG.9).

The absorbance of this solution was measured by spectramax M2 fromMolecular Devices in a quartz cuvette. FIG. 7 b shows the absorbancemeasured from the gold-coated and uncoated, Tm containing, α-NaYF₄nanophosphors with a maximum at around 525 nm as well as the uncoatednanophosphors. The Au/Tm(Er) ratio is the molar ratio of Au³⁺ and Tm³⁺(Er³⁺ ) in the reaction mixture. The absorbance increases with theconcentration of the 0.1% HAuCl₄ in the solution with Au/Tm ratio of0.57 corresponding to easily decipherable shell thickness of 4-8 nm.While the uncoated nanophosphors did not show any maximum due toabsorbance, a scattering indicated by exponential decay with increasingwavelength was observed. The observations were similar for Er containingnanophosphors. FIG. 10 a shows optical absorbance of 0.1% solutions ofcubic α-NaYF₄:20% Yb:2% Er with different gold coatings. FIG. 10 b showsup-conversion emission in the red when excited by 975 nm. FIG. 10 cshows up-conversion emission in the near infrared also upon excitationwith 975 nm.

Upconversion emission experiments were performed by a continuous wavelaser (Lasermate) operating at 975 nm. The PTG (Princeton instrumentscontrolled the shutter on the Princeton Instruments PI-MAX camera fittedwith a charge-coupled device sensor. The photons emitted by theupconverting NP were focused on to Acton spectrapro 300i seriesspectrometer by objective and condensing lenses. The controller, thecamera and the spectrometer were synchronized by Winview/32 softwareprovided by Princeton Instruments. Quartz cuvette was used in allexperiments and the laser with a power density of 67W/cm-2 was focusedon the quartz cuvette and the emission collected by a combination ofcondensing lens that focused the light on the slit of the spectrometer.

FIG. 11 a-c show the up-conversion emission characteristics of goldcoated and uncoated 0.1% solution of cubic α-NaYF₄:20% Yb:1% Tmnanophosphors excited at 975 nm. FIG. 11 d is an integrated area of theemission spectra from gold-coated phosphors normalized to the uncoatedphosphor. With increasing concentration of 0.1% HAuCl₄ in the reactionmixture, the blue (¹D₂→³F₄ and ¹G₄→³H₆), red (¹G₄→³F₄) and thenear-infrared (³H₄→³H₆) emission intensities shows a systematic increasewith no quenching effect attributable to the presence of completenanoshells of on top of the nanophosphors.15 The two different samplestested had an error less than 5%, which was the uncertainty in the poweroutput of the laser. Hence the error is represented as the percentage ofthe value. However, with Er doped nanophosphors, it appears that theplasmonic enhancement is limited to low concentrations of HAuCl4 andhence lower concentration of Au on the surface of the nanophosphor. FIG.12 a shows up-conversion emission from 0.1% solutions with differentgold coatings on cubic α-NaYF4:20% Yb:2% Er excited at 975 nm. FIG. 12 bis an integrated area of the emission spectra from gold-coated phosphorsnormalized to the uncoated phosphor. In Er containing nanophosphors(FIG. 12), with Au/Er ratio of 0.095, there is a two-fold increase inthe emission of green (²H_(11/2)→⁴I_(15/2) and ²S_(3/2)→⁴I_(15/2)) andred emission (⁴F_(9/2)→⁴Il_(5/2)) and suppression of the near-infraredemission (²S_(3/2)→4I_(13/2)) (FIG. 10), while for higher concentrationsthe of gold the intensities of emissions are comparable. The error barsin the plot correspond to the standard deviation from three differentsamples. This could be due to competing effect of the plasmonic fieldsand inner filter effect of the plasmonic shell that have absorbancearound 530 nm. It should be noted that the plasmonic enhancement ofup-conversion in an Er³⁺ containing NaYF₄ has been achieved by carefullyarranging NPs and plasmonic nanoparticles using atomic forcemicroscopy.[16]

It should be noted that the two sets of emission ¹G₄→³H₆, ¹G₄→³F₄ and²S_(3/2)→⁴I_(15/2), ²S_(3/2)→⁴I_(13/2) for Tm and Er, respectively havethe same initial state in their respective ions, but the gold shell hasquite different influence on the two emissions. In Tm containingnanophosphors, the plasmonic effect is positive with the enhancement ofboth the emissions, while for Er the higher energy transition,²S_(3/2)→⁴I_(15/2), is enhanced and the ²S_(3/2)→4I_(13/2) issuppressed, showcasing the subtleties of the plasmonic field effects onenergies of emissions. This interplay between emission and plasmonicshell suggests the probable direct coupling of the Plasmon electricfields to the energy level of the emission electron.

Example 3 Preparation of Labeled Gold-Coated CSNPs

SA and SA-TMR conjugate (from Invitrogen in Carlsbad, Calif.) isadsorbed onto gold-coated phosphor CSNPs. FIG. 13 provides anillustration of the labeling process. Gold-coated phosphor CSNPs wereprepared as described previously. 500 μl of 0.005% SA and SA-TMR in 10mM phosphate buffered silane (PBS) was reacted with 50 μl of 50 mM2-iminothiolane HCl (Traut's reagent). The reaction was facilitated bystirring and allowed to proceed under ambient conditions in a darkenvironment for two hours. Different volumes of 0.1% gold-coatedphosphor CSNP were introduced and stirred continuously for 10-12 h.

The effectiveness of binding was established by comparing the controlsolution with the supernatant obtained after isolating the gold-coatedphosphor CSNP from the reaction mixture in a FRET experiment. To thisend, control solutions of 0.005% SA and SA-TMR containing Traut'sreagent were suitably diluted by double distilled deionized water tomatch the different volumes of gold-coated phosphor CSNP solution. Theabsorbance was determined of the supernatant obtained after isolatingthe gold-coated phosphor CSNP conjugated with SA and SA-TMR bycentrifugation at 3500 rpm for 6 min. 600 ml of the solutions(supernatant and control) were taken and diluted to 1 ml before takingthe absorbance. For comparison, absorbance of the 0.1% gold-coatedphosphor CSNP without conjugation with SA or SA-TMR was also measured.The absorbance spectra is shown in FIG. 14 wherein the open circlesrepresent absorbance of the supernatant extracted after conjugation withgold-coated phosphor CSNP and the closed circles represent 0.003% SA-TMRconjugate in 6 mM PBS.

The inset of FIG. 14 shows percent conjugation of SA-TMR. This wascalculated by taking the absorbance of the control solutions at 520 nmto be 100. The percentage difference in the absorbance at 520 nm betweenthe supernatant and the control was estimated as the percent ofconjugation. As the amount of gold-coated phosphor CSNP was increased inthe solution, more SA-TMR was conjugated to gold-coated phosphor CSNP.

These SA-TMR conjugated gold-coated phosphor CSNP were further testedfor FRET by depositing the nanoparticles on a microscopic glass slide.Sample handling was designed to minimize photobleaching of the dye. Toeliminate any effect from SA, SA was conjugated to gold-coated phosphorCSNP and the phosphorescence compared with nascent gold-coated phosphorCSNP and SA-TMR conjugated to gold-coated phosphor CSNP upon excitationwith a 975 nm laser. The resulting spectrum is shown in FIG. 15, whereinthe squares represent emission from gold-coated phosphor CSNP, thediamonds represent emission from gold-coated phosphor CSNP conjugated toSA, and the triangles represent emission from the gold-coated phosphorCSNP conjugated to SA-TMR. It was observed that when the gold-coatedphosphor CSNP was conjugated to SA alone, the green emission (520.5 and540.5 nm) did not decrease. This was established by taking the ratio ofintensities of emissions at 540.5 and 520.5 nm (ratios of intensitiesfor the samples is shown in the inset of FIG. 15. On the other hand,when SA-TMR conjugated to gold-coated phosphor CSNP was excited with 975nm laser, the ratio was different. Considering the fact that the laserpower was very low and the fact that both nascent and SA-only conjugatedgold-coated phosphor CSNP have the same ratios, it is most likely thatthe change in the ratio is caused by the acceptance of the radiantenergy of gold-coated phosphor CSNP by TMR dye. The TMR fluorescencefrom the resonance energy transfer around 560 nm was not observed.

FRET experiments were performed with a PICOQUANT™ pulsed diode laseroperating at 975 nm and 80 MHz available from PicoQuant GmbH, Berlin,Germany. The photons emitted by the upconverting phosphor were focusedon to Acton SpectraPro 300i series spectrometer by objective andcondensing lenses, and imaged by Princeton Instruments PI-MAX camerafitted with a charge couple device sensor. Both the spectrometer andcamera are available from Princeton Instruments, Trenton, N.J.

In another embodiment, the fluorophore conjugated to SA is an ALEXAFLUORTM available from Invitrogen. The spectrum from gold-coatedphosphor CSNP conjugated to streptavidin with the ALEXA FLUOR™ uponexcitation at 660 nm is provided in FIG. 16.

Example 4 Gold-Coated CSNPs with Magnetic Component

Magnetic gold-coated phosphor CSNPs are also synthesized by the sol-gelcitrate technique described previously. The citrate route for magneticparticles avoids cumbersome synthetic protocols and worrisomeincompatible reagents in the solution. This is beneficial when using theresulting nanoparticles in a biomarker platform.

Magnetite gold-coated phosphor CSNPs were prepared by co-precipitationof Fe₃O₄ upon addition of NH₄OH solution to a mixture of FeCl₂ andFeCl₃, taken in a 1:2 molar ratio. The reaction consisted of twosteps—1) the precipitation step and 2) the capping step with citricacid. The reaction was performed under Argon flow at 80° C. withvigorous stirring of the solution. The precipitation reaction wasinitiated by introducing 40 mmoles of NH₄OH into 40 ml water containing4.3 mmoles of FeCl₂ and 8.6 mmoles of FeCl₃. The heating was continuedfor 30 min upon the addition of the NH₄OH. 5.2 mmoles of citric acid wasthen added and the heating continued at an increased temperature of 95°C. for 90 min. Thereafter, the resulting solution was diluted to 1000 mlto give a preferred concentration of 1% Fe₃O₄.

The magnetic particles in up-converting phosphors were derived from suchstock solutions mentioned above. The precursors used for up-convertingphosphors were water soluble salts of a lanthanide. The ratio ofconcentrations of the lanthanide, Fe₃O₄, sodium citrate and sodiumfluoride were optimized to incorporate the magnetic particles into thelanthanide matrix. In a typical reaction, 80 mmoles of Fe₃O₄ werereacted with 1.56 mmoles of Y(NO₃)₃, 0.66 mmoles of Yb(NO₃)₃ and 40micromoles of Er(NO₃)₃, amounting to 0.6 M of lanthanum nitrates. Equalvolume of 0.6 M sodium citrate and 12 ml of 1M solution of NaF wasintroduced to form the NaYF₄:Yb:Er matrix. The reaction mixture washeated at 90° C. for two hours.

To introduce the gold shell on top of the magnetic core phosphor, adilute solution of HAuCl4 was introduced to coat the dielectric surfacewith gold. This procedure is the same as described previously inreference to Examples 1 and 2.

FIGS. 17 a and 17 b are transmission electron micrographs showing 100 nmnanophosphor, NaYF₄:20% Yb:2% Er, with Fe₃O₄ core and gold shell. Thegold shell is visible as a 5 nm contour around the nanophosphor. Thesuper paramagnetic Fe₃O₄ nanoparticles are typically in the 10-15 nmrange.

FIGS. 18 a and 18 b are transmission electron micrographs showing the100 nm nanophosphor, NaYF₄:20% Yb:2% Er, with Fe₃O₄ core and without thegold shell.

FIG. 19 illustrates superparamagnetic hysteresis measurements of 100 nmnanophosphor, NaYF₄:20% Yb:2% Er, with Fe₃O₄ core and with/without goldshell from a Vibrating Sample Magnetometer. The magnetization curves areshown for various fractions of Fe₃O₄ in a 100 nm nanophosphor with 5 nmgold shell. For comparison, uncoated samples were also tested. Sample 32corresponds to the TEM images shown in FIG. 17, and sample 31 to theuncoated samples represented in FIG. 18.

Example 5 Two-Metal Coated Phosphor CSNP

A bi-metallic shell consisting of Au and Ag was prepared by employingAgNO3 in water.

In a typical reaction, the concentration of the AgNO3 solution was fixedat 0.05 M. The synthesis involved the addition of the AgNO3 solution tothe initially reduced gold on the surface of the phosphor.

In a typically reaction carried out at 90° C., 1 ml, 0.2M of rare earthchlorides were stirred with 2 ml, 0.2M of sodium citrate followed by theaddition of 4 ml, 1M sodium fluoride. Addition of the initial goldprecursor, 150 μl solution containing 380 nmoles HAuCl₄ was followed bythe addition of 2.5 μmoles of AgNO₃. Two more additions of 380 nmoles ofHAuCl₄ followed the addition of AgNO₃ at intervals of one hour. FIG. 20shows the absorbance spectra of three different solutions of NaYF₄:20%Yb:2% Er containing different molar ratios of Ag and Au.

Example 6 Au/An-Coated CSNPs as Biomarkers

The Au/Ag shell coated phosphor CSNPs can be labeled for use asbiomarkers. Labeled Au/Ag-coated phosphor CSNPs such as those preparedin Example 5 are useful as in vivo biomarkers. The Au/Ag coating is aninert platform to which various biological molecules can be attached,such as the SA and SA-TMR. The Au/Ag-coated phosphor CSNPs are excitedby a low energy laser which does not harm the biological moleculeattached and the surrounding tissues are transparent to this wavelengthand therefore the excitation laser does not harm surrounding tissues.Additionally, the low energy of the excitation source minimizesexcitation of surrounding molecules, such as proteins, therebyminimizing background fluorescence signals from those surroundingmolecules. The nanoparticles do not photo bleach leading to improvedsensitivity and reliability of data. The addition of Ag aids in theincorporation of many different fluorophores on the surface, which canbe selectively enhanced by the presence of the metal.

Example 7 Temperature Measurement with Au/An-Coated CSNPs

The plasmon resonance of the metal nanoparticle converts the lightenergy to heat energy (Alexander O. Govorov and Hugh H. Richardson,Nanotoday, 2, 30, 2007). This is observed as an increase in temperatureof the matrix inside and the surroundings. The increase in thetemperature of the inside matrix when the plasmonic metal is coated as ashell has a square dependence on the radius of the particle. The largerthe particle greater is the temperature raise. Also, the stronger theplasmon resonance the greater the increase in the temperature (Lee, J.,et al., Nano Lett. 4, 2323, 2004). It is possible to increase andharvest the increase in the temperature by coating gold with silver,which generates 10 times more heat energy than just gold. This allowsthe use of low power to achieve a certain temperature. Also, coating ofsilver will increase the possibility to achieve phase transformation inthe core and in the surroundings. For example, in the case of phosphorCSNP, the material NaYF₄ exists as a meta-stable cubic phase at lowtemperature. This phase has low up-conversion efficiency. Conversion ofthe metastable cubic phase to the hexagonal phase is achieved by heatingthe particles. With silver as one of the metal shell element, brightemissions are selectively achieved through local heating of the phosphorby subjecting the phosphor CSNP to plasmonic excitation. This will opennew possibilities with optical actuation and sensing.

The Au/Ag as well as the gold-coated phosphor CSNPs disclosed herein canbe used as temperature sensors. Due to the size and the inert nature ofthe metal shell, the nanoparticles can be used to determine temperaturein vivo. The CSNPs are placed in the desired location for determining atemperature and excited with a near infrared laser and the resultingemission is measured. For gold-coated phosphor CSNPs containing Er³⁺ andTm³⁺, the two emission peaks of interest occur at 520 and 540 nm. Fromthe ratio of the intensities of the emission at those two wavelengthstemperature is determined. Using spectroscopic term symbols, theelectronic excitation from the ground state 4I15/2 to the 4F7/2 statepopulates two lower states ²H_(11/2) and ⁴S_(3/2) based on the Boltzmanndistribution, which is influenced by the temperature of the particle.The emission intensities at 520 and 540 nm are then incorporated intothe following equation: log[Intensity(520)/Intensity(540)]=A+(B/T),where A and B are constants for Er³⁺ and Tm³⁺, and T is the absolutetemperature measured in degree Kelvin. Due to the increased heatgeneration with silver, a Au/Ag-coated CSNP is more useful fortemperature measurement than a gold-coated phosphor CSNP.

Gold-coated and Au/Ag-coated nanoparticle temperature sensors are usefulin monitoring temperature in microfluidic devices and studyingindividual cell thermogenics. Additionally, they could be used todetermine the denaturation temperature of single protein molecules andindividual double strand DNA hybridization temperatures.

Hyperthermic cancer therapy is another area where temperature monitoringin small spaces is relevant. There is currently a lack of preciseknowledge about the amount of heat delivered to the cancerous cells andhaving such knowledge is important to the efficacy of the therapy.Gold-coated or Au/Ag-coated phosphor CSNPs could be introduced to thesite of the tumor prior to the start of the hyperthermic therapy. Asheat is being applied, the gold-coated or Au/Ag-coated phosphor CSNPsare excited and their emission measured to keep the physician informedof the amount of heat actually present at the site of the therapy.

Example 8 Hyperthermic Cancer Therapy with Au/An-Coated CSNPs

In addition to temperature measurement in hyperthermic therapy,Au/Ag-coated phosphor CSNPs can act as heat actuator and thus deliverheat to the area where therapy is needed. There is a lack of safe andaccurate delivery and actuation of heat in vivo. Because Au/Ag-coatedphosphor CSNPs operate as temperature sensors, they can both deliver theheat and allow for monitoring of the resulting temperature.

Example 9 Au/An-Coated CSNPs as Anti-Counterfeiting Measure in SecurityPapers

Security paper is any paper that incorporates one or more features thatcan be used to identify the paper and therefore identify any item thatincorporates the security paper as an original. Examples of items thatincorporate security paper include, but are not limited to, currency,stock certificates, birth certificates, checks, academic certificates,passports, titles to property, lottery tickets, and legal documents.

Gold-coated phosphor CSNPs are an excellent feature to include insecurity paper. The gold-coated phosphor CSNPs have a reddish color thatis visible to the naked eye. Upon excitation with near-infrared laser,light is emitted. The emitted light can be in the red, blue or greenwavelengths. If light of multiple wavelengths is emitted, the emittedlight may appear to be a combination of red, blue or green to theobserver. For example, if the emitted light is in the red and bluewavelengths, it would appear purple to the observer. Near-infraredlasers can be handheld devices and are safe to operate. Therefore,gold-coated phosphor CSNPs are an excellent solution for authenticatingitems incorporating paper.

Adding silver to the shell to make a Au/Ag-coated phosphor CSNP addsfunctionality as more colors can be used.

The nanoparticles can be incorporated into the paper during itsmanufacture or after manufacture by printing them onto the paper Ink jetprinting is an excellent option for printing the nanoparticles in anink. The Au/Ag shell can have various modifications attached that wouldbe allow for tuning the properties of the ink to optimize theprinting—surface tension, viscosity, etc. Current ink jet printingtechnology allows for printing on nanoscale as reported by DuncanGraham-Rowe in MIT's Technology Review on Sep. 13, 2007. The printednanoparticles can therefore be so small as to require magnification todetect. Additionally, the nanoparticles can be printed in a pattern ortext and because of the nanoscale resolution, a very large amount ofinformation can be placed on the paper in a small space. Because eachrare earth element has a unique excitation wavelength, theauthentication technique can be tailored by using different rare earthelements in the upconverting phosphor core.

REFERENCES

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All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the invention as defined in the appended claims.

We claim:
 1. A nanoparticle comprising a phosphorescent core and a metalshell wherein said metal shell comprises a first and a second metal. 2.The nanoparticle of claim 1 wherein said phosphorescent core comprisesan upconverting phosphor.
 3. The nanoparticle of claim 1 wherein saidphosphorescent core comprises a trivalent rare earth cation.
 4. Thenanoparticle of claim 3 wherein said phosphorescent core furthercomprises a monovalent alkali metal.
 5. The nanoparticle of claim 3further comprising a second trivalent rare earth cation.
 6. Thenanoparticle of claim 5 further comprising a third trivalent rare earthcation.
 7. The nanoparticle of claim 3 wherein said trivalent rare earthcation is Tm³⁺.
 8. The nanoparticle of claim 3 wherein said trivalentrare earth cation is Er³⁺.
 9. The nanoparticle of claim 3 wherein saidtrivalent rare earth cation is Y³⁺.
 10. The nanoparticle of claim 3wherein said trivalent rare earth cation is Yb³⁺.
 11. The nanoparticleof claim 1 further comprising an anion selected from the groupconsisting of a Group 15, 16 and 17 anion.
 12. The nanoparticle of claim11 wherein said anion is a Group 17 anion.
 13. The nanoparticle of claim12 wherein said Group 17 anion is F⁻.
 14. The nanoparticle of claim 3wherein said monovalent alkali metal is Na⁺.
 15. The nanoparticle ofclaim 1 wherein said first and second metals are transition metals. 16.The nanoparticle of claim 1 wherein said first metal is selected fromthe group consisting of Au, Ag, Pt, Pd, Rh and Re.
 17. The nanoparticleof claim 16 wherein said second metal is selected from the groupconsisting of Au, Ag, Pt, Pd, Rh and Re.
 18. The nanoparticle of claim 1further comprising a fluorescent tag.
 19. The nanoparticle of claim 1further comprising a magnetic component.
 20. The nanoparticle of claim19 wherein said phosphorescent core comprises said magnetic component.21. The nanoparticle of claim 19 wherein said magnetic componentcomprises Fe₃O₄. 22-24. (canceled)